Method for the rapid detection of whole microorganisms on retaining membranes by use of chaotropic agents

ABSTRACT

The present invention relates to a method detection of microorganisms concentrated from body fluids as serum, sputum, pericardiac fluid, urine or other fluid on the surface of retaining membranes by specific partners of reaction that bind to antigenic components of the retained organisms is facilitated when the organisms in suspension in body fluids and/or concentrated on the membrane are treated with a high molar concentration of a chaotropic agent as guanidine, urea, isothiocyanate, thiourea.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. §371 of InternationalApplication No. PCT/EP01/00829, filed Jan. 26, 2001, designating theUnited States and published in English, which claims priority toEuropean Application No. 00870013.0 filed Feb. 1, 2000.

FIELD OF THE INVENTION

The present invention is in the field of diagnostic and is related to amethod and kit for the rapid detection of whole microorganisms onretaining membranes by the use of chaotropic agents.

BACKGROUND OF THE INVENTION

Detection of microorganisms in body fluids, water and alimentaryproducts allows diagnostic and prognostic of many infectious diseases.Cells and microorganisms needed for the making of vaccines demands asevere control of the sterility of the produced media, usually achievedby culturing an aliquot of the medium whose sterility one wants tocontrol, in a growth-promoting medium during several days. Said growthand identification of microorganisms are time consuming, difficult andfrequently takes two to three weeks before results are available.

For diagnostic purposes, such a method of determination relying onculture of blood sample or of sputum exists, when there is a need todiagnose the presence of mycobacterial entities. The time needed toarrive at a diagnostic conclusion is however very long, extendingsometimes to 4 weeks. For other microorganisms, such as the etiologicalagents of malaria (especially Plasmodium falciparum, leishmaniasis,gonorrhea, syphilis, tuberculosis, malaria, tick-borne Lyme disease, aswell as meningitis due to a variety of vectors such as Neisseria,Haemophilus, Streptococcus, Listeria and Mycobacterium, a rapiddiagnostic method putting the whole pathogen in evidence does not existand there is a need for such rapid method.

Rapid methods of detection of antigens and antibodies are now currentlyin use for antibodies and proteinic and glycoproteinic antigens whosesize is sufficiently small for them to be transported in a lateral flowchromatographic system. For larger entities (such as whole bacteria andeukaryotic organisms), this technique is inapplicable because theanalytes are too big to move with the flow. These microorganisms oflarge size are also able to condense on retaining membranes in such away that the sensitivity of their detection in situ is considerablyenhanced.

However, an intolerably high level of false positive or negative resultsis also observed. The phenomenon of immunological quenching induced bybody fluids is well known. This quenching prevents the recognition ofthe whole organisms by specific binding partners as antibodies andinduces false negative results.

STATE OF THE ART

The document EP-0306206 describes a diagnostic device comprising afunnel that channels the fluid under analysis onto a defined part of afilter membrane that retains the analyte. The funnel is thereafterremoved and the membrane is further treated by signal-generatingmaterials and other reagents as washing fluids, applied simultaneouslyto the test area and adjacent area of the membrane. The simultaneoustreatment of adjacent area supposedly provides a negative control toassist in detecting non-specific binding and thereby false-positiveresults. In said document, the sample is confined to a discrete area ofthe filter and the whole of the filter is then treated with reagent,thereby giving a true negative control. This document mentions water asfluid passing through the restricted aperture and omits to describe thetype of membranes used. Such an improved device relying on directdetermination of the presence of pathogens on the membrane is indeedapplicable only with water. More charged fluids such as serum,accumulated into a single discrete area of the filter tend to clog thisfilter up, prevent the easy flow-through of subsequently added fluids(such as a wash medium) and cause the succeeding washing fluids, appliedto the whole of the membrane, to flow around that area withoutpenetrating it. Further, even with water, the device is specificallydeveloped and meant to visualise false positive cases, therebyindicating that the occurrence of false positive cases is possible, evenwith water. No such device applied to detect whole microorganisms bydirect labelling is in use for body fluids such as serum, cerebrospinalfluid, pericardial fluid, urine or other body fluid.

The use of a chaotropic agent like sodium thiocyanate has been advocatedfor suspensions of microorganisms in water before their condensing on amembrane filter, but the concentration advocated is 0.04 molar and onlywater is recognised as a fluid suitable for treatment (documentJP-05034350). This teaching addresses the issue of detection ofmicroorganisms in water, whose clumping may be prevented or deliquescedinto singly suspended organisms with the help of low concentrations ofdisrupting agents.

The increase in sensitivity of an immunochemical assay of collagen hasbeen claimed in the presence of a chaotropic agent. Neither urea, norguanidine are mentioned as suitable, but thiocyanate used at aconcentration of 0.1 to 1.6 molar included in the reaction mix is(document WO92/16846). This teaching addresses the issue of collagensolubilised in an assay medium containing a chaotropic agent asisocyanate and recognised by a binding partner.

Similarly, a treatment of a sample with various detergents and achaotropic agent as urea prior to immunoassay has been claimed to beapplicable to blood samples investigated by immunoassay for the presenceof viruses as hepatitis C virus (document WO99/06836). In this case, thechaotropic agent is contacted together with different surfactants withthe sample under analysis, not as a washing solution, and no attempt ismade to detect in a direct way the pathogenic entity condensed on thesurface of a membrane, but a lateral flow immunoassay is used in thedetection of the analyte present in the treated biological fluid.

The document U.S. Pat. No. 5,714,343 describes a device consisting inabsorbing pads surmounted by a retaining membrane. The fluid is passedthrough the retaining membrane and the microorganisms potentiallyretained on the membrane are visualised by a chromogenic agent having anoxidation potential such that the reagent can be reduced by microbialdehydrogenase, yielding a visibly coloured product indicative of thepresence of microorganisms in the sample. The retaining membrane havingpores (0.75 to 1.2 μm) larger than the dehydrogenase-activemicroorganisms it is supposed to retain, the capture of the bacteria onsuch filters is achieved by mechanical retention. The reason for thisunlogically great pore-size used at the risk to let a large amount ofthe analyte pass through the filter, is the possible occurrence of falsepositive cases with smaller pores, since other reducing compounds thanbacteria (e.g. free reducing enzymes, ascorbic acid, glutathione) maythen, be retained, yielding false positive results. Further, althoughthe device is said to be useful for water, blood, milk and urineanalysis, only physiological water is described in examples 1 to 7.Theprincipal advantage of this method is the signal-generating material,consisting in a chromogen that forms a visible precipitate of formozaneafter reaction with bacterial dehydrogenase. Growth of the bacteria invitro for several days after collection on the retaining membrane istherewith avoided, and much time saved. Many bacteria and yeasts as wellas leukocytes possess a dehydrogenase activity and the detecting methoddoes not discriminate between them, except for the selective destructionof dehydrogenase synthesised by gram positive bacteria, leaving thedehydrogenase from gram-negative bacteria still active. This selectivedestruction of dehydrogenase produced by gram-positive bacteria isachieved by either incubation of the sample with octyl glucoside, whichsuppresses the dehydrogenase activity of all gram+ bacteria tested, orelse 0.5 M guanidine, that suppresses the dehydrogenase activity of onlysome gram+bacteria while at least one gram-bacterium, Pseudomonas, isalso affected (only living bacteria possessing active dehydrogenase).

Therefore, one of the drawbacks of said technique is that it does notallow a specific detection, because a large number of microorganismspossessing active dehydrogenase and other reducting enzymes are detectedand identified.

The documents U.S. Pat. Nos. 4,695,537 and 4,808,518 underline the factthat hypertonic solutions of agents as sodium chloride, potassiumthiocyanate, guanidine and others may be applied to antigens insuspension and to intracellularly located antigens, at concentrationsbetween 1.0 and 5.0 M, preferably between 2.0 and 3.5 M. Theseconcentrations do not reduce the antigenic properties of the antigen butimprove the yield.

The document EP 0 234 941 indicates that treatment of viral subunitswith denaturing concentrations of guanidine (from 5 to 8 molar) (afterthe purification of the virus and its disruption into subunits withdetergents) improves the purity of these subunits for the adsorption ofantibodies in an Elisa. It is known that denaturing concentrations ofchaotropic agents destroy protein assemblies into subunits, as is thecase with the alpha and beta subunits of human chorionic gonadotropin.Sometimes, as is the case with the subunits of HCG, the immunogenicityof the subunits is not affected by this hypertonic treatment.Tuberculoproteins are isolated by treating tubercular material withchaotropic agents (guanidine, urea and phenol) which reduce the tuberclepathogen to immunogenic tuberculoproteins (FR-2082226). However, thesedocuments do not suggest the maintenance of the pathogen under analysiswhole and intact, without any reduction into subunits, so that itremains of a size sufficient to be mechanically retained on a filteringmembrane and thereafter its direct detection.

AIMS OF THE INVENTION

The present invention is related to a method and kit for the rapiddetection of whole microorganisms, which do not present or reduce thepossible false positive or false negative results affecting the methodof the state of the art.

SUMMARY OF THE INVENTION

A simple detection system able to demonstrate in a direct way, in asensitive way and in a specific way the presence of whole pathogensbelonging to the bacterial kingdom or else eukaryotic parasites asPlasmodium falciparum or toxoplasma in various body fluids as serum,cerebrospinal fluid, pericardial fluid etc. condensed on a retainingmembrane, is not yet available, although such a detection would greatlyimprove the rapid diagnostic of several pathogens. Such a system must,per force, exploit the specific binding capacities existing amongspecific binding partners for the microorganisms one wishes to detect,as specific antibodies, protein A, protein G.

The occurrence of false positive results are due to contaminatingsubstances present in the body fluids wherein the microorganisms aresuspended and quenching induced by them, which adsorbs to the surface ofthese microorganisms and prevent their recognition by specific bindingpartners (either when these organisms are in suspension in these bodyfluids or after, these microorganisms have been condensed out of thesefluids, on the surface of retaining membranes).

The present invention is related to a method for the detection of one ormore microorganisms present in a liquid sample, preferably a biologicalliquid sample, said method comprising at least the following steps:

-   (a) possibly adding to said liquid sample a reagent solution    comprising a chaotropic substance having preferably a concentration    able to rupture hydrogen bonds;-   (b) filtering the liquid sample through a filter having a pore size    which is small enough to prevent passage of microorganisms through    the filter, but large enough to permit passage of any soluble    material present in the sample, whereby the microorganisms are    retained on the filter;-   (c) possibly passing said reagent solution comprising a chaotropic    substance through the filter having the retained microorganisms    thereon, said chaotropic agent solution having preferably a    concentration able to rupture (suppress) hydrogen bonds, and-   (d) passing one or several labelled reagents (signal-generating    reagents) through the filter, at least one of said reagents being    specific for the microorganisms to be detected.

The chactropic agent solution comprises urea, guanidine, thiourea,isothiocyanate or a mixture thereof and the concentration of thechaotropic agent in the solution is higher than 2M, preferably between 4and 8M (denaturating concentration).

The microorganisms could be present in any biological sample, preferablyin a charged biological sample such as sputum, blood, serum, plasma,cephalo-rachidian fluid or urine obtained from any animal patient,including the human and are preferably pathogenic bacteria or eukaryoticparasites detected either simultaneously or successively.

Contaminants of beverages and foods, as listeria, yeasts and molds arealso detectable. The type of filter used to retain the analytes understudy is usually cellulose, paper, nitrocellulose or nylon, or othertypes selected by the man skilled in the art.

The specific labelled reagents used for the detection of microorganismsare preferably antibodies, possibly coupled directly or indirectly to amarker, such as gold micellae, biotine, enzymes, chromophores, stainedlatex beads, enzymes, fluorophores, radioactive compounds or a mixturethereof.

The present invention is related also to a kit comprising means andmedia for performing the detection method according to the invention,said kit comprising filtering membranes of predetermined pore size toretain specific microorganisms, one or several chaotropic agents, one orseveral labelled reagents (signal-generating reagents), one of thembeing specific for the monitored microorganisms.

If necessary, the method and kit could be adapted to allow the rapiddetection of microorganisms present in a sample by using an automate andmeans for performing automatically the specific detection according tothe invention.

DETAILED DESCRIPTION OF THE INVENTION

The presence of microorganisms as the Koch's bacillus (Mycobacteriumtuberculosis), Plasmodium falciparum, or other bacteria and parasitesdifficult to diagnose, as leishmaniasis, gonorrhea, syphilis, tick-borneLyme disease, as well as meningitis due to a variety of vectors such asNeisseria, Haemophilus, Streptococcus, and Listeria, and yeasts andworms, is detected in human and animal serum and other body fluids bycondensing the microorganisms present in these fluids on the surface ofa membrane that allows the passage of the fluid, but retains themicroorganisms on its surface. Thereafter by washing the membrane toeliminate contaminants, treating the membrane with a chaotropic agent asguanidine, urea, guanidine isothiocyanate or similar agent, at aconcentration high enough to clean the microorganisms of all thequenching elements that prevent their recognition by specific bindingpartners, treat the retaining membrane and the microorganisms condensedon its surface with a binding partner specific for the microorganism,such as antibodies, thereafter one washes the membrane free of excessbinding reagent and label the antibodies bound to the microorganism witha binding partner specific for the class of gammaglobulins of the animalspecies in which antibodies against the microorganism were raised, e.g.rabbit, goat, donkey, as antibodies against rabbit or goat or donkeygammaglobulins, protein A or protein Gj, said binding partner beingitself coupled to a marker (i.e. an agent susceptible to amplify thelabelling, as colloidal gold or end enzyme).

The detection of microorganisms having the size of bacteria or bigger,(spirochetes, trypanosomes and worms) present-in body fluids as serumand urine obtained by means of specific binding partners as antibodies,protein A or protein G is not easy.

However microorganisms in solution or condensed on the surface of amembrane resist the disruptive action of high molar concentrations ofchaotropic agents as guanidine, urea, thiocyanate, thiourea,isothiocyanate, perchlorate etc. while becoming readily accessibletherewith to recognition by specific binding partners. The samemicroorganisms condensed from the same sera on the same retainingmembranes and treated with the same chaotropic agents used at a molaritytoo low to exploit their disruptive potential on proteins (as forexample 0.5 molar guanidine used to destroy the activity ofgram+bacterial dehydrogenase) are not recognised by their specificbinding partners. Therefore a tenfold higher concentration of thechaotropic agents is unexpectedly beneficial in the conduct of animmunoassay.

Molarities of urea and guanidine ranging from about 4 to 8 are needed tohave an effect. With other chaotropic agents, as thiourea andisocyanate, lower concentrations ranging between 2 and 5 molar, areadequate.

High molar concentrations of chaotropic agents as guanidine and ureahave been for a long time applied, mostly together with organic solventsas phenol and chloroform, to liberate the nucleic acids DNA and RNA fromeukaryotic cells, from bacterial cells as Escherichia coli and fromvirus entities. The fact that nucleic acid is obtained followingtreatment of cells and viruses with chaotropic agents indicates that thecell membranes, the nuclear membranes and tertiary structures ofproteins in viruses are destroyed by this treatment. This effectprecludes any evident application of chaotropic agents at high molarconcentrations for the purpose of the present invention.

Chaotropic agents as urea and guanidine applied at concentrationsranging between 4 to 8 molar, useful and necessary for the exploitationof their protein denaturing properties and their ability to suppresshydrogen bonds, are currently used either to eliminate proteins fromnucleic acid preparations or else to separate protein subunits,thereafter isolated by separation methods based on the differentphysico-chemical properties of the separated products. For example, thealpha and beta subunits of chorionic gonadotropin are separated in thepresence of 8 molar urea at 40° C. and the two subunits are subsequentlyisolated by passage on a DEAE-cellulose chromatography column. Purifiedtuberculoproteins are isolated by submitting tubercular material to aprecipitation stage at an acid pH, carried out in the presence of asubstance capable of rupturing hydrogen bonds, as urea, guanidine,formamide, phenol, etc. The precipitated material is thereaftercontacted with a modified cellulose bearing a basic group such as DEAE(see document FR-2082226). In these cases, the molarity of thechaotropic substance is sufficiently high to exploit its disruptingproperties on substances kept in solution, with the purpose toeffectuate a separation and solubilisation of the various substancescontained within the original preparation.

The main problem encountered in the development of assays aiming atdetecting in a direct way pathogens condensed on a retaining membrane isthe quenching induced by charged liquid media as serum, quenching ofsuch a nature that the microorganism is not anymore readily recognisedby specific binding partners. Many sera induce a quenching thatconsiderably reduces the signal that is detected after application ofsignal-producing elements. More particularly, the pathogen isolated fromsome sera and condensed at the surface of the retaining membrane doesnot react to antibodies directed against it, which precludes thepossibility to thereafter visualise the presence of these antibodies bysignal-amplifying binding partners as gold-labelled protein A or asperoxidase-labelled antibodies specifically binding with gammaglobulins.Whereas some sera and less charged fluids as urine, culture media andwater are satisfactory in this sense, the fact that some sera inducefalse-negative results precludes the routine use of such methods for thedetermination of the presence of pathogens.

Quite unexpectedly, it was discovered that high molar concentrations ofchaotropic agents as guanidine, urea, thiourea, isocyanate, applieddirectly on the liquid sample under analysis and/or on the retainingmembrane following the condensing of the microorganism on its surface,greatly facilitates the reaction of specific binding partners with themicroorganism under analysis. A device consisting in a cartridgecontaining absorbent pads, topped with a retaining membrane, iscurrently available for the making of such assays as described in thedocument U.S. Pat. No. 5,714,343 incorporated herein by reference. Usingsuch a device, the following experiments were done, which may serve asexamples.

EXAMPLE 1

500 μl of human serum spiked with tuberculous bacillus was passed over amembrane whose mean pore size was 0.45 microns. This pore size is smallenough to retain the great majority of the bacilli, whose mean size is0.6×4 μm. After passage of the liquid sample, the retaining membrane waswashed with 200 μl of washing medium. The membrane was further washedwith 200 μl of a 6 molar solution of guanidine hydrochloride at pH 8.0.A 200 μl wash with washing medium eliminated the quanidine and themembrane was thereafter treated with 100 μl of a dilute solution ofrabbit antibodies against mycobacterium tuberculosis. The antibodieswere obtained in rabbits repeatedly inoculated with heat-killedMycobacterium tuberculosis (Difco). After a 200 μl wash, 100.μl ofgold-labelled protein A at a concentration of A₅₂₀=0.75 were applied,with a positive result consisting in a red-pink spot in the middle ofthe membrane.

The same analysis performed in the absence of a chaotropic reagentproduced a diffuse barely coloured spot instead.

The same analysis performed with a solution of guanidine whoseconcentration was lowered to about 4.5 moles yielded a spot whoseintensity was similar to the positive control (6 molar guanidine).

The same analysis performed with a solution of guanidine whoseconcentration was increased to 7 moles yielded a spot whose intensitywas similar to that obtained with about 6 molar concentration inguanidine.

EXAMPLE 2

An analysis identical to that described in Example 1 was performed witha solution that was 7 molar in urea. The same positive result as inexample 1 was obtained, whereas concentrations lower than about 4.5molar were ineffective in revealing a satisfactory signal. Aconcentration of urea superior to about 8 molar reduced the signal.

EXAMPLE 3

The same experiment as in Example 1 was conducted, with the guanidinewash solution brought to pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0,12.0 and 13.0 with concentrated HCl or 10 molar NaOH. The solutions ofthe chaotropic agent that were of an acidity equal or inferior to pH 6.0were much less satisfactory that those whose pH was above neutrality. Ata basic pH 13.0, the signal was reduced. Optimal signal delivery wasobtained with a solution of guanidine at pH 9.0.

EXAMPLE 4

The same experiment as in example 1 was conducted with the chaotropicagents 4 molar potassium thiocyanate, 2 molar thiourea, 6 molarguanidine containing 2 molar thiourea, 3 molar urea mixed with 3 molarguanidine. Solubilisation difficulties due to the presence of varioussurfactants currently used in immunoassays and known from the man of theart did not allow higher molarities, concentrations for thiocyanate andthiourea than those here applied. These chaotropic agents were notsuperior to urea or quanidine used alone.

EXAMPLE 5

These experiments showed that, when no sputum is used, a membrane with apore size 0.45 μm has a satisfactory retention power. However, whensputum is used, the 0.45 μm pore sized membrane tends to clog up easier.A membrane with a larger pore size is preferable. To further favourflow, a glass fiber pre-filter is needed, that retains undissolvedmatter and viscous matter present in sputum.

Sputum from a TB tuberculosis patient was collected at the Pneumologydepartment of the University Hospital of Strasbourg (France). The sputumunderwent regular analysis and was found to contain TB bacilli by thebacilloscopy technique based on the Ziehl-Nielsen Stain. The sputum wasfluidised by addition of 0.25% N-acetyl-cysteine and 1% NaOH (finalconcentrations). This fluidising method is standard in mycobacteriologyfor the treatment of sputum before its use in cultures. The NaOH isadded as a fluidisation medium and also in order to decontaminate thesample and kill all other bacteria present, before culture for tuberclebacilli is initiated. The NaOH is not indispensable for the liquefactionand N-acetyl-cysteine, at concentrations between 0.5% and 2% andalkaline pH, is able to liquefy sputum in two minutes. Other methods offluidisation, based on hypochlorite or sodium dodecyl sulphate, werefound to work just as well. In particular, bleach may be appropriate forlow income countries because it is cheap, available locally and itbetter respects the integrity of the retaining membrane than NaOH. Analteration of the retaining membrane, made of nitro-cellulose, wasobserved when the sample fluid was treated with NaOH at 1%. Aneutralisation of the fluid with HCl was needed before passing it on anitro-cellulose retaining membrane. Other means of fluidisation (bleach,dithiothreitol) proved compatible with the retaining membranes used. Anyfluidising agent that respects the antigenicity of the bacteria and doesnot alter the retaining membrane is satisfactory.

A fiber-glass prefilter pad was placed on top of the nitrocelluloseretaining membrane, so as to retain the unsolubilised material remainingin the fluidised sputum. 200 μl of fluidised sputum were passed throughthe prefilter and the 0.8 μnitrocellulose membrane, before thefiberglass prefilter was removed. The retaining membrane was washed with2 times 100 μl of 0.2M phosphate pH 7.2 containing 0.2% Tween 20.Afterthe wash, the membrane was treated with 6 M guanidine, and furthercontacted with a rabbit antiserum against M. tuberculosis appropriatelydiluted 1:160 in phosphate-buffered saline pH 7.2, containing 0.2% Tween20.After a washing step, the signal present on the membrane amplifiedwith a reagent consisting in 30 nm gold-sensitised rabbit anti-goat IgG.The sensitivity of the system detects about 3000 mycobacterialentities/ml.

EXAMPLE 6

The detection of mycobacteria in sputum was done as in example 5, with amembrane whose pore size was 0.45 μm. This small pore size was foundacceptable although it retarded the flow rate, but a slower flow of thereagents enhanced the sensitivity of the method by allowing a longercontact and prolonged reaction time of the reagents with the antigens.Rabbit anti-mycobacterial serum was used to label the antigen retainedon the membrane. The secondary partner of reaction was composed ofantibodies against rabbit IgG raised in donkeys. The enhancer was 30nm-gold-labelled Protein G., which reacts well with equine antibodies.The sensitivity reached was about 3000 entities/ml.

EXAMPLE 7

A membrane with a pore-size of 0.45 μm was used to collect themycobacteria suspended in sputum in an experiment applied as in example5.After labelling the mycobacteria present on the membrane with specificrabbit antibodies, a secondary binding partner consisting of ProteinA-20 nm colloidal gold, was applied (Sigma Chemical Co, St Louis, Mo.).The results were similar to those obtained in example 6, when thesecondary antibody was gold-sensitised goat anti-rabbit IgG, i.e. 3000particles/ml.

EXAMPLE 8

Human infections caused by Chlamydia trachomatis primarily involve theeyes and the genital tract. The morphology of the chlamydiae resolvesinto Elementary Bodies (0.2 to 0.4 μm) that can be detected byimmunofluorescence or enzyme linked immunosorbent assays and ReticulateBodies which is the intracellular, metabolically active form (0.6 to 1.0μm), rarely observed microscopically The laboratory diagnosis is basedon a variety of methods (complement fixation, microimmunofluorescence,enzyme linked immunosorbent assays, direct microscopic examination,isolation of the microorganisms and nucleic acid techniques). Forgenital tract infections, specimens for assay are urethral and cervicalswabs, and urine, with the aim to detect the etiological agent.

Human urine was spiked with decreasing amounts of the pathogen Chlamydiatrachomatis Elementary bodies. Antibodies raised in rabbits were used tolabel the pathogen. One and a half millilitres of the spiked urine werepassed on a filter with mean more size 0.2 μm, that was able to retainthe pathogen on its surface. No prefilter was useful. Revelation of thepresence of the pathogen was done by treating the membrane first with 5M guanidine at pH 8.0, thereafter with the specific antibodies, followedwith Gold-protein A, that reacts with rabbit gammaglobulins. In a secondanalysis, antibodies raised in goats were used. Gold-labelled protein Gwas used to reveal the presence of the goat antibodies and an enhancerconsisting in Gold-labelled pig gammaglobulins was finally applied. Thesensitivity of this detection system was compared to that obtained bythe rapid test chlamydia, a commercial diagnostic kit (Abbott). Whereasa single amplification system (rabbit antibodies and Gold-protein A)yielded similar results, the use of an enhancer (Gold-labelled pigantibodies) increased the sensitivity two-fold.

EXAMPLE 9

Toxoplasmosis is caused by Toxoplasma gondii, a sporozoan whoseindividual cells are 4 to 7 μm long. Diagnosis relies essentially ondetection of specific antibodies in serum but the sites most commonlyattacked are the lymph nodes, brain, eyes and lungs. Direct examinationof sputum, vaginal exudates, spinal, pleural and peritoneal fluids arepossible, but rarely practised. No diagnostic kits exist for directexamination of sputum and body fluids.

Toxoplasma gondii organisms were mixed with sputum, the sputum wasliquefied and 1 ml passed on an 0.8 μm membrane fitted with a prefilter.The same procedure as in example 7 was followed. The results wereexcellent, with the detection of about 200 pathogenic entities in thesample.

EXAMPLE 10

Few diagnostic kits for the detection of Neisseria gonorrhoeae areavailable. The laboratory diagnosis of gonococcal infection is basedprimarily on the identification of the etiologic agent by microscopicexamination and by culture. Cultures derived from sterile sites(cerebrospinal fluid, blood, synovial fluid) usually provide adefinitive diagnostic but positive cultures from non-sterile sites areof uncertain value. No serologic test is commercially available yet.

Sputum was spiked with known concentrations of Neisseria gonorrhoeaeantigen, as in example 5.The pore-size of the retaining membrane was0.45 μm, compatible with the size of the organisms (0.6 to 1.0 μindiameter). The solubilisation of the sputum was done with 0.2% Sodiumdodecyl sulphate at pH 9.5, treatment that was found not to alter theimmunoreactivity of the pathogen. The presence of N.gonorrhoeae-specific rabbit IgG on the surface of the membrane,indicative of the presence of the pathogen, was further pursued as perexample 7, with similar results.

EXAMPLE 11

The microscopic observation of the adult schizonts of Plasmodiumfalciparum in blood smears is the laboratory diagnostic of malaria.

Human fresh whole blood was spiked with formalin-inactivated schizonts.After the spiking, the blood was hemolysed with 1% Nonidet P 40 (finalconcentration) and 500 μl of the fluid was passed on a glass prefilterand collected on a retaining membrane with pore size 0.8 μm. Guanidineat a 4 molar concentration at pH 8.0 was found satisfactory in thisanalysis. A high concentration of 8 molar urea was also found adequatebut this remarkable resistance of the schizonts may have been obtainedby their fixation with formalin. The mouse antibodies used to label theantigen were revealed with Gold-labelled Protein G. Distinct red spotson the membrane signalled the presence of individual organisms.

A great number of possibilities were investigated, using eitherantibodies labelled with peroxidase or with gold, using protein A andprotein G labelled with gold or with peroxidase, and the usefulness ofsecondary amplification steps was also investigated. The most reliableand easy method was found to be the one described in example 7: 500 μlto 1.5 ml (depending on the proteinic load of the processed sample) ofsample is passed through a retaining membrane. The organisms presumablyconcentrated on the surface of the membrane are then treated with asolution 6 molar in guanidine hydrochloride at pH 8.After a wash, themembrane is treated with rabbit antibodies against the analyte and thepresence of the antibodies potentially attached to the antigenicdeterminants of the analyte are put in evidence with gold-labelledprotein A.

EXAMPLE 12

A treatment of the analysed sample directly with high concentrations ofa chaotropic agent, before the concentration of the analyte on theretaining surface of a filtering membrane, possibly followed by awashing with the chaotropic agent, yields the best results.

100 μl of sputum found positive for TB by a microscopic examination weresolubilised with 100 μl of solubilising solution consisting of 5%N-acetylcysteine and 0.5% mercaptoethanol in 1% NaOH brought at pH 12.This solubilising solution is standard procedure but other solubilisingmethods (e.g. sodium hyposulphite) known by the person skilled in theart are equally applicable.

After the sputum has been digested (5 minutes at room temperature), thesample is mixed with 1.3 ml of a solution 7 molar in guanidine at pH 8.5containing 0.01% Tween 20, and processed as per example 7.

A red central spot is observed after completion of the test, notobservable when negative samples are processed in an identical manner.

EXAMPLE 13

100 μl of sputum found positive for TB by a microscopic analysis wasmixed with 100 μl of a solution that was 7 molar in guanidine at pH12.00. After solubilisation of the sputum, 100 μl of NaClO at 12° wasadded to the mixture and digestion of the sputum was pursued during 15minutes at Room Temperature. The sample was thereafter mixed with 1.3 mlof a solution that was 7 molar in guanidine at pH 8.5 and processed asper example 12.

A red central spot is observed after completion of the test, notobservable when negative samples are processed in an identical manner.

1. A method for the detection of one of several types of microorganismsin a liquid sample suspected of containing microorganisms, comprisingthe steps of: (a) adding to said liquid sample a reagent solutioncomprising a chaotropic substance having a concentration between about 4and about 8 M to produce a mixture; (b) filtering said mixture through afilter having a pore size which is small enough to prevent passage ofmicroorganisms through the filter but large enough to permit passage ofsoluble material present in said mixture, whereby any microorganisms areretained on the filter, and (c) passing one or more labelled reagentsthrough the filter to produce labeled microorganisms, wherein at leastone of said reagents is specific for the microorganisms to be detected,and (d) detecting said labeled microorganisms.
 2. The method accordingto claim 1, wherein the pH of the reagent solution comprising thechaotropic agent is at a pH of between about 6.0 and about
 12. 3. Themethod according to claim 2, wherein the pH of the reagent solutioncomprising the chaotropic agent is about pH 9.0.
 4. The method accordingto claim 1, wherein said liquid sample is obtained from an animal orhuman patient and is selected from the group consisting of sputum,blood, serum, plasma, cephalo-rachidian fluid, pericardial fluid,cerebrospinal fluid, vaginal exudates, spinal fluid, pleural fluid,peritoneal fluid, synovial fluid and urine.
 5. The method according toclaim 1, wherein said microorganisms are pathogenic bacteria orpathogenic eukaryotic parasites.
 6. The method according to claim 1,wherein said microorganisms are detected simultaneously or successively.7. The method according to claim 1, wherein said filter is a membranemade of nitrocellulose or nylon.
 8. The method according to claim 1,wherein said chaotropic substance is selected from the group consistingof urea, guanidine, thiourea, isothiocyanate and a mixture thereof. 9.The method according to claim 1, wherein the labelled reagents specificfor the microorganisms to be detected are antibodies.
 10. A kit for thedetection of one or more microorganisms in a sample suspected ofcontaining microorganisms, comprising a filter having a pore size whichis small enough to prevent passage of microorganisms through the filterbut large enough to permit passage of any soluble material in thesample, one or several chaotropic substances in solution wherein the oneor several chaotropic substances have a concentration between about 4and about 8 M, and one or several labeling reagents, wherein said one orseveral labeling reagents are antibodies specific for the microorganismsto be detected.
 11. The kit according to claim 10, wherein said one orseveral chaotropic substances are selected from the group consisting ofurea, guanidine, thiourea, isothiocyanate and a mixture thereof.
 12. Themethod according to claim 9, wherein said antibodies are coupleddirectly or indirectly to a marker.
 13. The method according to claim12, wherein said marker is selected from the group consisting of gold,micellae, enzymes, chromophores, stained latex beads, fluorophores, anda mixture thereof.
 14. A method for the detection of one or severaltypes of microorganisms in a liquid sample suspected of containingmicroorganisms, comprising the steps of: (a) filtering the liquid samplethrough a filter having a pore size which is small enough to preventpassage of microorganisms through the filter but large enough to permitpassage of any soluble material present in the sample, whereby themicroorganisms are retained on the filter; (b) passing a reagentsolution comprising a chaotropic substance through the filter having anymicroorganisms retained thereon, said chaotropic reagent solution havinga concentration between about 4 and about 8 M; and (c) passing one ormore labelled reagents through the filter having thechaotropic-contacted microorganisms retained thereon, to producelabelled chaotropic-contacted microorganisms, wherein at least one ofsaid reagents is specific for the microorganisms to be detected, and (d)detecting said labeled microorganisms.
 15. The method according to claim14, wherein the pH of the reagent solution comprising the chaotropicreagent is at a pH of between about 6.0 and about
 12. 16. The methodaccording to claim 15, wherein the pH of the reagent solution comprisingthe chaotropic reagent is about pH 9.0.
 17. The method according toclaim 16, wherein said liquid sample is obtained from an animal or humanpatient and is selected from the group consisting of sputum, blood,serum, plasma, cephalo-rachidian fluid, pericardial fluid, cerebrospinalfluid, vaginal exudates, spinal fluid, pleural fluid, peritoneal fluid,synovial fluid and urine.
 18. The method according to claim 16, whereinsaid microorganisms are pathogenic bacteria or pathogenic eukaryoticparasites.
 19. The method according to claim 16, wherein saidmicroorganisms are detected simultaneously or successively.
 20. Themethod according to claim 16, wherein said filter is a membrane made ofnitrocellulose or nylon.
 21. The method according to claim 16, whereinsaid chaotropic substance is selected from the group consisting of urea,guanidine, thiourea, isothiocyanate and a mixture thereof.
 22. Themethod according to claim 16, wherein the labelled reagents specific forthe microorganisms to be detected are antibodies.
 23. The methodaccording to claim 22, wherein said antibodies are coupled directly orindirectly to a marker.
 24. The method according to claim 23, whereinsaid marker is selected from the group consisting of gold, micellae,enzymes, chromophores, stained latex beads, fluorophores, and a mixturethereof.